In biological and chemical analyses, the use of analyte molecules labelled with reporter groups is routine. This invention addresses the idea of providing reagents having at least two analyte groups linked to one or more reporter groups. Such reagents can be used, in ways described below, to generate much more analytical information than can simple labelled analytes. It is possible to code reporter groups so that reagents carrying multiple analyte groups and multiple reporter groups can by synthesised combinatorially and used simultaneously and the reporter groups resolved in the analytical stage.
WO 93/06121 (Affymax) describes a synthetic oligomer library comprising a plurality of different members, each member comprising an oligomer composed of a sequence of monomers linked to one or more identifier tags identifying the sequence of monomers in the oligomer. The linkage between the oligomer and the identifier tag preferably comprises a solid particle. The identifier tag is preferably an oligonucleotide.
Proc. Natl. Acad Sci., Vol 89, No. 12, Jun. 15, 1992, pages 5381-5383 (S Brenner and R A Lerner) describe encoded combinatorial chemistry for making a library of reagents each containing a genetic oligonucleotide tag.
In Rapid Communications in Mass Spectrometry, Vol 6, pages 369-372 (1992), G R Parr et al describe matrix-assisted laser desorption/ionisation mass spectrometry of synthetic oligodeoxyribonucleotides.
In Nucleic Acids Research, Vol 21, No. 15, Jul. 25, 1993, pages 3347-3357, E Nordhoff et al describe the ion stability of nucleic acids in infra-red matrix-assisted laser desorption/ionisation mass spectrometry.
In one aspect the present invention provides a reagent comprising
a) an analyte moiety comprising at least two analyte residues, and linked to
b) a tag moiety comprising one or more reporter groups suitable for detection by mass spectrometry, excluding oligonucleotides,
wherein a reporter group designates an analyte residue, and the reporter group at each position of the tag moiety is chosen to designate an analyte residue at a defined position of the analyte moiety.
Preferably the analyte moiety is linked to the tag moiety by a link which is cleavable, e.g. photocleavable. There may be provided a linker group to which the analyte moiety and the tag moiety are both attached. Preferably the analyte moiety is a chain of n analyte residues, and the tag moiety is a chain of up to n reporter groups, the reporter group at each position of the tag chain being chosen to designate the analyte residue at a corresponding position of the analyte chain. n is an integer of at least 2, preferably 3 to 20.
The invention may be used for the detection of all analytes of interest. These include, but are not limited to, a protein/peptide chain so that the analyte residues are amino acid residues; a nucleic acid/oligonucleotide chain so that the analyte residues are nucleotide residues; a carbohydrate chain so that the analyte residues are sugar residues. Additionally the analyte may be a class of small molecules with biological, pharmacological or therapeutic activity. For example it could be a core molecule with the ability to vary various substituent groups eg. alkyl, esters, amines, ethers etc. in a combinatorial manner with mass spectrometry tags.
The tag moiety and/or the or each reporter group in it is capable of being observed/detected/analysed so as to provide information about the nature of the analyte moiety, and/or the analyte residues in it.
In one embodiment, the reagent has the formula A-L-R where A is a chain of n analyte residues constituting the analyte moiety, L is the linker, R is a chain of up to n reporter groups constituting the tag moiety, and n is 2-20, wherein the tag moiety contains information defining the location of analyte residues in the analyte moiety.
The tag moiety consists of one or more reporter groups distinguishable by mass and thus capable of being analysed by mass spectrometry. The reporter groups may be chemically different and thus distinguished from one another by molecular weight. Or the reporter groups may be chemically identical, but distinguished from one another by containing different isotopes (e.g. 12C/13C and 1H/2H as discussed below). The tag moiety is, and/or the reporter groups are, suitable or adapted for analysis by mass spectrometry e.g. after cleavage by photochemical or other means from the reagent.
The advantages of mass spectrometry as a detection system are: its great sensitivityxe2x80x94only a few hundred molecules are needed to give a good signal; its wide dynamic range and high resolving powerxe2x80x94molecules in the mass range 100 to 200,000 Daltons can be resolved with a resolution better than 0.01; its versatilityxe2x80x94molecules of many different chemical structures are readily analysed; the potential to image analytes by combining mass spectrometry with, for example, scanning laser desorption: and the ability to make quantitative as opposed to merely qualitative measurements.
Thus mass-labelling combines advantages of radioactivity and fluorescence and has additional attributes which suggest novel applications.
In another aspect, the invention provides a library of the above reagents, wherein the library consists of a plurality of reagents each comprising a different analyte moiety of n analyte residues. For example, the library may consist of 4n reagents each comprising a different oligonucleotide chain of n nucleotide residues. The reagents of the library may be present mixed together in solution.
In another aspect, the invention provides an assay method which comprises the steps of: providing a target substance; incubating the target substance with the said library of reagents under conditions to cause at least one reagent to bind to the target substance; removing non-bound reagents; recovering the tag moieties of the or each bound reagent; and analysing the recovered tag moieties as an indication of the nature of the analyte moieties bound to the target substance.
The target substance may be immobilised, as this provides a convenient means for separating bound from non-bound reagent. In one aspect, the target substance may be an organism or tissue or group of cells, and the assay may be performed to screen a family of candidate drugs. In another aspect, the target substance may be a nucleic acid, and this aspect is discussed in greater detail below.